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  • 16S Metagenomics Kit for Ion Torrent

    Hello,
    Has anyone in this forum tried out the 16S Metagenomics Kit for Ion Torrent and then if so, did you try to analyze the data with Qiime?

    I previously had analyzed Ion Torrent data using Qiime and the Brazilian Microbiome Project pipeline:

    however this kit makes use of 7 primers and sequencing 7 variable regions.
    • 16S primer set V2-4-8 (300 µL)
    • 16S primer set V3-6,7-9 (300 µL)

    So my previous pipeline won't quite work with this. Has anyone tried this and used Qiime for the analysis?

    Life Tech delivers a software pipeline along with this for the analysis but I am curious to find someone that has tried it out and was able to get good results?


    thanks,
    Jen

  • #2
    Hey Jen,

    A colleague and myself are working on trying the kit out and comparisons between it's workflow and Qiime. I'll let you know our experience as we make progress.

    Best Regards,

    Nabeeh

    Comment


    • #3
      Hi Nabeh,
      How far along have you gotten? I am just curious about what you might do with the qiime pipeline given that you do not know the primer sequences for the 6 different primers used in the kit? Have you thought about that yet?
      Thanks,
      Jen

      Comment


      • #4
        Hello Jen,

        I thought the Qiime workflow has an option to use datasets composed of reads from multiple regions of the 16s sequence, something analogous to the method implemented in Emirge and Emirge2, but I might be mistaken.

        I've did a few initial analyses with the IonReporter. We're quantifying all the result numbers (Valid Reads, Ignored Reads, Reads Mapped, etc.) to better understand what the Ion 16s metagenomics workflow is actually doing with the data. So what I can say is that you have a few search options using the IonReporter: i) search against the LifeTech MicroSEQ curated database of ~15K full-length 16s rRNA sequences; ii) search against the Greengenes 16s rRNA database of ~400K full-length 16s rRNA sequence; iii) search using a two-step where it first searches reads against the MicroSEQ and searches reads that don't match MicroSEQ against the Greengenes database. It gives you OTUs based on a % identity threshold you specify when starting the workflow. Using default parameters for reads, as I understand, >97% identity results in genus level identifications; >99% identity results in species level identifications. It specifies slash calls as when reads have a tie in % identity range for two or more OTUs at a particular level (i.e. reads with 97% identity for both Mycobacterium and Nocardia). It compiles all the taxa represented across the different variable regions and estimates a consensus identification list of all the OTU identified with the different V region sequences detected in the sample. It doesn't appear that the IonReporter is trying to assemble the reads in any sort of way as is done with other workflows.

        If people are looking for IonReporter to be a potential replacement for Qiime, SILVA, etc., then this is not the case. Those are capable of quite a bit more indepth analyses that are not part of IonReporter at this point.

        My labmate and I are working this inbetween grant and paper edits, so progress is admittedly slow. I'll try to comeback as I find any new stuff.

        Best Regards,

        Nabeeh

        Comment


        • #5
          Hello Nabeeh,
          Have you made any progress with this kit?
          Jennifer

          Comment


          • #6
            Hello Jennifer,

            Is there something specifically that you are interested in? I have made some progress, although not as much as I had hoped, so I might not be able to answer you question(s). I have a conference call with ThermoFisher about the 16S Metagenomics kit later today. As the kit and IonReporter are currently configured, you must analyze the data in a different workflow (Qiime) to really make interesting/relevant comparisons between samples.

            The kit is pretty flexible. There are two reaction pools that amplify different sets of the V regions of the 16s, so you can amplify a subset or all depending on if that is necessary for your question(s) of interest. The lab side of the kit ends up being laborious in comparison to going with an adapted 16s V4 analysis on the PGM. I'd much rather set up several sets of PCR, pool then do a single cleanup as opposed to a 2 PCRs, pooling then going through ligating barcodes and all the purification steps done in the Ion library construction. Also, the recommendation of read numbers per sample is really dependent upon your sample complexity and question in mind. Our clinical samples aren't that diverse depending on what taxonomic level of resolution you desire (family, genus vs. species), so we find that you can analyze more samples and use fewer reads than are recommended by ThermoFisher.

            Best Regards,

            Nabeeh

            Comment


            • #7
              Hey Guys,

              Sorry to interrupt but I’m a bit desperate to get advice on this kit. I’m attempting to determine the microbial diversity in an environmental sample and was thinking of using the 16S kit with Ion Torrent. The kit is quite new at the sequence facility I use and thus I’m hesitating to venture into the unknown. Would you guys recommend the kit? And would Illumina MiSeq be a better option to Ion Torrent PGM?

              I’m a newbie in the sequencing world so I’m hoping you guys can help.

              Thanks in advance,

              Jade

              Comment


              • #8
                Greetings Jade,

                It depends on the needs of your experimental design and if you were doing this yourself. If you are looking for species identification instead of genus and family level identifications of taxa, then the 16S Metagenomics Kit is your option. How many environmental samples/replicates per sample are you going to run? The protocol is very hands on/laborious compared to the standard microbiome sequencing, so it gets more cumbersome as you increase sample numbers that are common in microbiome experiments today. Also, what are you trying to do after identification of species in the sample? Comparing samples is something that is not included in the current IonReporter Metagenomics workflow.

                The IonReporter workflow has two databases they analyze output against: the ThermoFisher MICROSEQ 16S Database that ThermoFisher manually curates, or the GreenGenes 16S Database. You can email ThermoFisher to check if certain groups of bacteria are included within the MICROSEQ database, but they are not willing to share those proprietary 16S sequences otherwise. They are also not willing to share the 16S primers used for the different V regions, so analyzing the data in other workflows (i.e. Qiime) is not as optimal as it could be.

                The output of the run includes a graph visualizing the proprotions of different taxanomic levels within a sample and a sample specific table with % identity and read counts for taxonimic units (i.e. Family, Genus, Species, etc.) identified within a given sample. The table is not in biom format and requires some manipulation to get into the format for scripts included in Qiime. So doing analyses where you are comparing lots or samples will require some additional bioinformatic tool development on your end to do comparative analyses at this point.

                For now, our lab mostly focuses on our custom V4 16S PGM sequencing and analyzing the data using Qiime. But if we are really trying to discriminate species in our clinical samples, we recommend the ThermoFisher 16S Metagenomics Kit.

                You can try to have a few samples sequenced on a 314 or 316 chip for a pretty low cost. Microbiome sequencing doesn't need that many reads per sample (~200K per sample is the recommendation from ThermoFisher, but that always depends on how diverse your samples are.

                I'm sorry if I didn't address what you were looking for, so feel free to ask specific questions.

                Best Regards,

                Nabeeh

                Comment


                • #9
                  Hello Jade,
                  Did you end up making a decision to use the 16s kit or did you go with something else? If you did use the kit, I was just wondering how your progress with it is coming along?

                  Thanks,
                  Jen

                  Comment


                  • #10
                    Hey Jen,

                    I have asked around for advice and most of the replies are similar to Nabeeh's but seeing that the kit is quite new none of the labs in my part of the world has tested it out yet. I rather decided to go with a workflow another research group have used using custom V4 primers. If I was more experienced in this type of research I would've tried the kit out.

                    Regards,
                    Jade

                    Comment


                    • #11
                      Ion 16S Metagenomic Kit

                      Dear all,

                      We have been using the Ion 16S Metagenomic kit since the beginning of the year. We have used the kit numerous times on mock bacterial communities and have been able to successfully identify all the microbial populations in our mock communities. On the 316 v2 chip we typically get between 65-80% loading with 58-68% usable reads. This enables us to multiplex 3-4 samples together with coverage depths >400x. We have also used the kit to perform human oral microbiome profiling with great success. However, it should be noted that the library prep steps are quite labor intensive and may not be the best option for diagnostic applications. We currently use the fusion method for our library preps using V1-2, V4, and V7-8 fused primer sets. We continue to use the Ion Reporter 16S metagenomic workflow for our analysis, because frankly my bioinformatics skills are rather limited and the Ion Reporter workflow is very intuitive and user friendly. We edited the default parameters to cater to our unique workflow after consulting with some bioinformatics specialists from Life Tech.
                      In summary, I would say if you are not too worried about turn around time or extensive lab work then the 16S metagenomic kit works very well, but for diagnostic labs I would advise you employ the the fusion approach to circumvent the hassles of adapter ligation and repetitive purification steps.

                      Comment


                      • #12
                        Hello,
                        I'm working on a metagenomic project and we choose to use the Ion 16S Metagenomics kit. I have the opportunity to work with the AB Library Builder System, which simplify the library construction.
                        Did anyone already work with it for the metagenomic library construction (in particular with the Ion Plus Fragment Kit?)
                        Thanks,

                        Sylvain

                        Comment

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